The proposed work involves continued studies into the hormonal control of carbohydrate metabolism in rat hepatocytes with particular emphasis on the regulation of hepatic pyruvate kinase by glucagon and epinephrine. Four specific objectives are planned. Particular emphasis will be given towards completing studies into quantification of the phosphorylation state of pyruvate kinase in hepatocytes treated with glucagon, epinephrine, and insulin. We also will complete studies into the differential actions of glucagon and epinephrine on carbohydrate metabolism in hepatocytes from fed and fasted rats and examine the changes seen as the rat matures. Studies into the influence of various buffers and metal ions on liver pyruvate kinase activity will be completed and experimens into control of the phosphorylation-dephosphorylation of the enzyme by cAMP dependent and cAMP independent protein kinases and phosphatases will be initiated. Finally, new studies concerning the influence of glucagon and insulin on the synthesis and turnover of pyruvate kinase in the isolated rat hepatocyte will be initiated. These latter studies will be done using a m nospecific antibody against rat liver pyruvate kinase which we have raised in goats.